PROJECT SUMMARY The Apicomplexa are a large phylum of intracellular pathogens that cause substantial disease in humans and animals worldwide. Their ability to infect their hosts, survive in their intracellular niche, and cause disease is strictly dependent on a series of unique organelles that are common to this group of pathogens. As most of the protein constituents of these organelles are not present in their human hosts, they represent intense areas of investigation in the search for new drug targets. Toxoplasma gondii has served as a model system for the study of apicomplexan parasites due of its ease of genetic manipulation and extensive set of molecular tools that have been developed for its study. While these molecular tools have focused on dissecting function at the individual gene level, the next big advances will require new approaches that are better able to evaluate precisely how proteins interact in complexes and how they participate in networks within the parasite to enable infections. To develop new tools that function at the protein level, we have been developing Toxoplasma strains with an expanded genetic code that can incorporate unnatural amino acids (UAAs) to study protein- protein interactions in vivo. This strategy uses an amber stop codon suppression system that enables strains to incorporate a photoreactive UAA at specific sites into a bait protein, which crosslink to binding partners when activated by UV light. We have demonstrated that parasites engineered to express an orthogonal amber suppressor tRNA and aminoacyl-tRNA synthetase pair efficiently incorporate the photoactivatable UAA p- azidophenylalanine (Azi) into control proteins and have also shown that we can obtain robust photoreactive crosslinking using this approach. In this application, we will engineer our system to enable incorporation of a second photoreactive UAA, p-benzoylphenylalanine. We will then utilize these systems to determine the precise interactions of the TgARO complex, which is critical for the function of the secretory rhoptries, as well as the interactions of a newly discovered protein in the conoid, which is important for invasion. Thus, this application develops new tools for protein-protein interaction studies in Toxoplasma and applies these tools to the study of critical protein complexes in the parasite.